The glucagon-like peptide-1 receptor agonist liraglutide improves hypoxia-induced pulmonary hypertension in mice partly via normalization of reduced ET(B) receptor expression.

The glucagon-like peptide-1 receptor (GLP-1R) agonist liraglutide is an incretin hormone mimetic used in the treatment of diabetes. However, the effects of liraglutide on pulmonary hypertension (PH) and pulmonary endothelin (ET) system are unknown. Eight-week-old C57BL6/J mice were injected liraglutide or vehicle for 5 weeks. One week after injection, the mice were exposed to either room air (normoxia) or chronic hypoxia (10 % O(2)) for 4 weeks. The right ventricular systolic pressure (RVSP) was significantly higher in hypoxia + vehicle group than in normoxia + vehicle group. ET-1 mRNA expression in the lungs was comparable among all the groups. ET(B) mRNA and protein expression in the lungs was significantly lower in hypoxia + vehicle group than in normoxia + vehicle group. The above changes were normalized by liraglutide treatment. The expression of phospho-eNOS and phospho-AMPK proteins in the lungs was significantly higher in hypoxia + liraglutide group than in normoxia + vehicle group. We demonstrated for the first time that liraglutide effectively improved RVSP and RV hypertrophy in hypoxia-induced PH mice by activating eNOS through normalization of impaired ET(B) pathway and augmentation of AMPK pathway. Therefore, GLP-1R agonists can be promising therapeutic agents for PH.

GABAA receptor-mediated input change on orexin neurons following sleep deprivation in mice.

Orexins are bioactive peptides, which have been shown to play a pivotal role in vigilance state transitions: the loss of orexin-producing neurons (orexin neurons) leads to narcolepsy with cataplexy in the human. However, the effect of the need for sleep (i.e., sleep pressure) on orexin neurons remains largely unknown. Here, we found that immunostaining intensities of the α1 subunit of the GABAA receptor and neuroligin 2, which is involved in inhibitory synapse specialization, on orexin neurons of mouse brain were significantly increased by 6-h sleep deprivation. In contrast, we noted that immunostaining intensities of the α2, γ2, and ß2/3 subunits of the GABAA receptor and Huntingtin-associated protein 1, which is involved in GABAAR trafficking, were not changed by 6-h sleep deprivation. Using a slice patch recording, orexin neurons demonstrated increased sensitivity to a GABAA receptor agonist together with synaptic plasticity changes after sleep deprivation when compared with an ad lib sleep condition. In summary, the GABAergic input property of orexin neurons responds rapidly to sleep deprivation. This molecular response of orexin neurons may thus play a role in the changes that accompany the need for sleep following prolonged wakefulness, in particular the decreased probability of a transition to wakefulness once recovery sleep has begun.

Multifocal granulomatous jejunitis associated with an argyrophilic gram-positive segmented filamentous bacterium in a Holstein cow.

Multifocal, raised, ulcerated firm nodules accompanied by an intussuscepted area were detected in the jejunum of an 8-year-old Holstein cow. The cut surfaces of the nodules were yellow-white. Microscopically, the lamina propria was expanded by an intense infiltration of epithelioid cells, multinucleate giant cells, macrophages, lymphocytes and neutrophils. Numerous bacteria were found within the granulomatous lesions. These were argyrophilic, gram-positive, periodic acid-Schiff-positive, segmented, rarely branched, elongate filamentous bacteria (2-28 µm in length, 0.2-0.35 µm in diameter). Ultrastructurally, a cell wall with an electron-transparent zone was detected. The present pathogen was clearly different from the argyrophilic, gram-negative, non-segmented, filamentous bacterium previously reported in a Holstein cow with jejunal granuloma. Comparative 16S rDNA gene sequencing analysis revealed that the organism was an unpublished species (GenBank accession number AB539875). This is the first report of bovine jejunal granuloma associated with an argyrophilic gram-positive segmented filamentous bacterium.

Lawsonia intracellularis and virulent Rhodococcus equi infection in a thoroughbred colt.

A 26-month-old thoroughbred colt with a 4-month history of continuous diarrhoea and weight loss was subject to necropsy examination. The small intestinal mucosa was thickened and this change particularly affected the terminal ileum. Microscopical examination revealed multifocal epithelial hyperplasia, with multifocal granulomas and marked lymphocytic infiltration of the lamina propria. Numerous gram-negative argyrophilic curved bacilli were observed within the cytoplasm of affected enterocytes. Macrophages and epithelioid cells forming the granulomas had abundant, lightly eosinophilic, foamy cytoplasm, with occasional large, clear vacuoles containing gram-positive coccobacilli. Immunohistochemical studies suggested that the argyrophilic bacilli were Lawsonia intracellularis and the gram-positive coccobacilli were Rhodococcus equi. L. intracellularis-specific DNA fragments were amplified from the affected ileocaecal mucosa by polymerase chain reaction. Virulent R. equi (VapA positive) was isolated in pure culture from the liver and mesenteric lymph nodes. These results suggested that the two intracytoplasmic organisms had induced multifocal proliferative and granulomatous enteritis accompanied by severe and extensive lymphocytic infiltration.

Endothelin-1 production is enhanced by rotenone, a mitochondrial complex I inhibitor, in cultured rat cardiomyocytes.

In chronic heart failure and acute myocardial infarction, the tissue level of endothelin (ET)-1 in the heart, as well as its plasma level, has been reported to increase markedly. There is, however, little information about what in these pathologic conditions leads to increased production of ET-1, and which type of cell in the heart produces ET-1. We examined the mRNA and peptide expression of ET-1 using cultured rat neonatal cardiomyocytes, in which mitochondrial dysfunction was induced by rotenone, a mitochondrial respiratory chain complex I inhibitor, because one of the common features in failing or ischemic hearts is an alteration in energy metabolism due to mitochondrial dysfunction. Rotenone increased glucose use by the culture cells within 12 h of addition without affecting cell viability, and depressed the mitochondrial membrane potential after 72 h, indicating the induction of mitochondrial dysfunction in cardiomyocytes. Rotenone induced significant increase in the expression level of mRNA for ET-1 within 1 h of addition. In accordance with this finding, immunoreactive ET-1 in culture medium increased 3 times after 24 h of incubation, suggesting active secretion of ET-1 from cultured cells treated with rotenone. Immunocytochemical analysis verified significant increase of ET-1 peptide in cardiomyocytes, confirming the production of ET-1 by cardiomyocytes. These results suggest that derangement of mitochondrial function in cardiomyocytes itself could lead to the increased production of ET-1 in cardiomyocytes, and that this mechanism may contribute to the increased production of ET-1 in failing and ischemic hearts.

Novel molecular mechanism of increased myocardial endothelin-1 expression in the failing heart involving the transcriptional factor hypoxia-inducible factor-1alpha induced for impaired myocardial energy metabolism.

BACKGROUND: Hypoxia-inducible factor (HIF)-1alpha is an important transcriptional factor that activates the gene expression of glycolytic enzymes, which are activated as compensation for impaired beta-oxidation of fatty acid in the failing heart. We reported that cardiac endothelin (ET)-1 expression is markedly increased in heart failure. The mechanism, however, is unknown. Because we found an HIF-1alpha binding site in the 5'-promoter region of the ET-1 gene, we hypothesized that HIF-1alpha is involved in this mechanism. METHODS AND RESULTS: In rat cardiomyocytes, luciferase assay and electrophoretic mobility shift assay showed that HIF-1alpha transcriptionally activates ET-1 gene expression by direct interaction with the predicted DNA binding site in the 5'-promoter region. HIF-1alpha mRNA and ET-1 mRNA in the failing heart increased during the aggravation of heart failure in vivo in animal models, ie, rats with myocardial infarction and hamsters with cardiomyopathy. In cultured cardiomyocytes treated with a mitochondrial inhibitor, HIF-1alpha mRNA and ET-1 mRNA were markedly increased with activated glycolysis, and antisense oligonucleotide for HIF-1alpha largely inhibited the increased gene expression of ET-1. CONCLUSIONS: The present study revealed a novel molecular mechanism of upregulation of myocardial ET-1 in heart failure, indicating that induction of HIF-1alpha to stimulate glycolysis as an adaptation in heart failure against impaired energy metabolism alternatively causes an elevation of cardiac ET-1 gene expression as a maladaptation.

Impairment of cardiac energy metabolism in vivo causes hemodynamic abnormality and increases cardiac expression of preproendothelin-1 mRNA.

We investigated whether impairment of myocardial energy metabolism attenuates cardiac function and increases cardiac endothelin-1 (ET-1) gene expression in rats. Three weeks after commencing administration of cobalt chloride (CoCl2), an inhibitor of mitochondrial function, the peak positive first derivative of left ventricular (LV) pressure, an indicator of myocardial contractility, was significantly decreased in the CoCl2-treated rats. LV end-diastolic pressure and right ventricular systolic pressure were increased in the CoCl2-treated rats. Echocardiography showed that fractional shortening was significantly decreased in the CoCl2-treated rats. Myocardial expressions of acyl-CoA synthase mRNA, an enzyme involved in fatty acid utilization, was markedly decreased in the CoCl2-treated rats. Under such conditions, myocardial expression of preproendothelin-1 mRNA and atrial natriuretic peptide (ANP) mRNA, molecular markers of heart failure, was markedly increased in the CoCl2 rats. In conclusion, the data suggest that impairment of myocardial energy metabolism causes hemodynamic abnormality and increases molecular markers of heart failure (ET-1, ANP mRNA). These data suggest that myocardial energy metabolism is one of the factors involved in the upregulation of ET-1 gene expression in the failing heart.

Mitochondrial dysfunction of cardiomyocytes causing impairment of cellular energy metabolism induces apoptosis, and concomitant increase in cardiac endothelin-1 expression.

It has been reported that at the end stage, apoptosis is involved in the progression of heart failure. It is suggested that cardiac energy metabolism is impaired during the progression of heart failure. Although the mechanism of induction of apoptosis in the failing heart varies according to the model of heart failure, it is not known whether an impairment of energy metabolism in cardiomyocytes is a primary cause of apoptosis. In this study, we applied mitochondrial inhibitors, such as rotenone, cobalt chloride and antimycin A, which inhibit mitochondrial function at different sites of the mitochondrial respiratory chain, to cardiomyocytes. All these reagents markedly decreased 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay (MTT) reduction activity, an indicator of mitochondrial function, of cardiomyocytes and greatly increased glucose consumption, suggesting that cardiac energy metabolism is switched from beta-oxidation of fatty acid to glycolysis. It was shown that after 48-72 h of treatment with each reagent, apoptosis was shown to occur by DNA laddering and increase in caspase activity. Interestingly, each reagent with a different action site greatly activated caspase-3, but not caspase-8 activity, suggesting that mitochondria are involved in induction of apoptosis. On the other hand, within 24 h of the treatment, when apoptosis of cardiomyocytes was not observed, the treated cardiomyocytes showed a marked increase in preproendothelin-1 and atrial natriuretic peptide (ANP) gene expressions. In conclusion, the present study suggests that mitochondrial dysfunction with impaired energy metabolism elevates gene expression of cardiac ET-1, an aggravating factor in heart failure, and then finally induces apoptosis in cardiomyocytes. The finding of marked increases in expression of molecular markers (ET-1 mRNA and ANP mRNA) in the failing heart, followed by apoptosis in the treated cardiomyocytes suggests that the inhibition of mitochondrial function of cultured cardiomyocytes provides a possible new in vitro model of heart failure.

Mitochondrial dysfunction increases expression of endothelin-1 and induces apoptosis through caspase-3 activation in rat cardiomyocytes in vitro.

We have reported that the expression of endothelin-1 (ET-1) increases in the failing heart. With the progress of heart failure, it has been reported that energy metabolism switches from mitochondrial b-oxidation to glycolysis. Furthermore, it has been reported that apoptosis is induced in the failing heart. However, it is not known how the gene expression of preproendothelin-1 and cellular apoptosis are affected by the mitochondrial dysfunction. Therefore, in order to elucidate this problem, we developed an in vitro model of mitochondrial dysfunction using rotenone, a mitochondrial respiratory chain complex I inhibitor, and studied preproendothelin-1 gene expression and apoptosis. Rotenone greatly increased the gene expression of pre-proendothelin-1 in cardiomyocytes. This result suggests that the gene expression of preproendothelin-1 is induced by the mitochondrial dysfunction. Furthermore, treatment of cardiomyocytes with rotenone induced an elevation of caspase-3 activity, and caused a marked increase in DNA laddering, an indication of apoptosis. In conclusion, it is suggested that mitochondrial impairment in primary cultured cardiomyocytes induced by rotenone in vitro, mimics some of the pathophysiological features of heart failure in vivo, and that ET-1 may have a role in myocardial dysfunction with impairment of mitochondria in the failing heart.

Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli production of soluble apoenzyme and in vitro formation of active holoenzyme.

Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli BL21(DE3) was accomplished by introducing a derivative of pET-23a(+) containing a copy of the coding gene into the multicloning site. E. coli BL21(DE3)/pETMNCAT produced abundant quantities of manganese catalase as insoluble inclusion bodies. Regeneration of active catalase was achieved by denaturation in guanidine hydrochloride and subsequent dialysis in the presence of manganese ion. When the E. coli chaperone genes GroEL, GroES, DnaK, DnaJ and GrpE were coexpressed with manganese catalase, a significant fraction of the overproduced protein was partitioned into the soluble fraction. However, almost all of the soluble enzyme was isolated in a manganese-deficient apo form which could subsequently be converted into active holoenzyme by incubation with manganese ion at high temperatures. Further experiments on this apo catalase suggested that the structure of this protein was virtually identical to the active holoenzyme.

Is the growth of brown adipocytes necessary for cold acclimation in mice?

Chronic exposure to cold increases the growth of brown adipose tissue and the resistance to more severe cold, thus improving thermogenesis. The present study examined the possibility that dietary compounds can modify cold acclimation. Adenosine (ADO) or adenine (ADE) were administered in drinking water (0.05%, w/v) for 5 weeks to male ddY strain mice from 4 weeks of age. At 5 weeks of age, the mice were exposed to 4 degrees C for 4 weeks. After termination of this period, the interscapular brown adipose tissue (IBAT) and rectal temperature on acute exposure to severe cold (-20 degrees C) for 60 min were measured. Chronic exposure to cold increased the weight of IBAT and made mice resistant to a fall in rectal temperature on exposure to severe cold. The growth of IBAT and improvement in thermogenesis can be used as a cold acclimation profile in ddY mice. The growth of IBAT was selectively prevented by ingestion of ADO. The improvement in thermogenesis was reduced, but only a little, by the ingestion of either ADO or ADE. Thus, growth of brown adipocytes might not be necessary for cold acclimation in mice.

Purification and cloning of a thermostable manganese catalase from a thermophilic bacterium.

We have purified a heat-stable catalase from a thermophilic bacterium, Thermus species strain YS 8-13. The enzyme was purified 160-fold from crude cellular extracts and possessed a specific activity of 8000 units/mg at 65 degrees C. The purified enzyme displayed the highest activity at pH 7 to 10 and temperatures around 85 degrees C. The catalase was determined to be a manganese catalase, based on results from atomic absorption spectra and inhibition experiments using sodium azide. The enzyme was composed of six identical subunits of molecular weight 36,000. Amino acid sequences determined from the purified protein were used to design oligonucleotide primers, which were in turn used to clone the coding gene. The nucleotide sequence of a 1.4-kb fragment of Thermus sp. YS 8-13 genomic DNA containing a 909-bp open reading frame was determined. The gene encoded a 302-residue polypeptide of deduced molecular weight 33,303. The deduced amino acid sequence displayed a region-specific homology with the sequences of the manganese catalase from a mesophilic organism, Lactobacillus plantarum.

[Effects of phenylalanine and tyrosine on cold acclimation in mice].

The effects of phenylalanine (PHE) and tyrosine (TYR) on cold acclimation were studied in mice of the ddY strain. At 5 weeks of age, mice were maintained at 4 degrees C for 4 weeks. Test groups of mice were supplied with a 0.05% (w/v) solution of PHE or TYR as drinking water in addition to water. We measured changes in body weight, intake of water and food, rectal temperature upon acute exposure to -20 degrees C, weight of interscapular brown adipose tissue (BAT) and the levels of glucose, nonesterified fatty acids (NEFA) and 3H-butyrate in the blood prior to and after exposure to the lower temperature of -20 degrees C. Chronic exposure to cold (4 degrees C) reduced body-weight gain for the first two weeks but weight gain recovered within the next two weeks. PHE and TYR partially inhibited the gain in body weight under exposing to cold. TYR reduced the gain of body weight under room temperature. Exposure to cold stimulated the daily consumption of food. Both PHE and TYR somewhat enhanced the food intake when exposed to cold. Exposure to cold rendered mice resistant to severe cold (-20 degrees C). Both PHE and TYR did not reduce this resistance except in the early state in the case of TYR. Exposure to cold increased the weight of BAT but both PHE and TYR prevented this increase. The effect of TYR was determined in the case fed under room temperature. Cold exposure changed the utilization of glucose, NEFA and 3H-butyrate in the blood when exposed to severe cold (-20 degrees C). Both PHE and TYR prevented the change in NEFA. It remains to be confirmed whether the growth of brown adipocytes is always necessary when mice acclimate to cold.

[Effects of adenosine and adenine on cold acclimation in mice].

The effects of adenosine (ADO) and adenine (ADE) on cold acclimation were studied in mice of the ddY strain. At 5 weeks of age, mice were maintained at 4 degrees C for 4 weeks. Test groups of mice were supplied with a 0.05% (w/v) solution of ADO or ADE as drinking water in addition to water. We measured changes in body weight, intake of water and food, rectal temperature upon acute exposure to -20 degrees C, weight of interscapular brown adipose tissue (BAT) and the levels of glucose, nonesterified fatty acids (NEFA) and 3H-butyrate in the blood prior to and after exposure to the lower temperature of -20 degrees C. Chronic exposure to cold (4 degrees C) reduced body-weight gain for the first two weeks but weight gain recovered within the next two weeks. ADO partially but selectively inhibited the gain in body weight. This effect was marked in mice maintained at room temperature. Such an inhibitory effect was the case of ADE in the room temperature. Exposure to cold stimulated the daily consumption of food. ADO further and selectively enhanced food intake, but ADE enhanced that in room temperature. Cold increased in daily intake of water. Both ADO and ADE accelerated the intake of water. This effect was marked in mice maintained at 4 degrees C and the effect of ADE was considerable. Mice chose to drink the water that contained ADO, while the choice of water that contained ADE was apparent only during exposure to cold. Exposure to cold rendered mice resistant to severe cold (-20 degrees C). ADO and ADE partially reduced this resistance. Exposure to cold increased the weight of BAT but ADO selectively prevented this increase. Cold did not change the levels of the various compounds measured in the blood. In mice exposed to 4 degrees C for 4 weeks, acute exposure to severe cold decreased the glucose level and increased the levels of NEFA and 3H-butyrate. ADO selectively prevented the changes in the levels of NEFA and 3H-butyrate. It remains to be confirmed whether the growth of brown adipocytes is always necessary when mice acclimate to cold.